This project will examine global protein expression changes in oral microbial communities and the community response to model host cells. We will select as model organisms the periodontal pathogen P. gingivalis, along with F. nucleatum and S. gordonii as representative of organism commonly found in dental biofilms and capable of synergistic interactions with P. gingivalis. In addition to differential protein expression as these organisms assemble into communities, we will also determine changes in protein expression induced by contact with gingival epithelial cells. The long-term goals of the study involve improving our fundamental understanding at the molecular level of events surrounding heterotypic oral biofilm formation and the interaction of these biofilms with host cells. Specific Aim 1 defines reference proteomes of P. gingivalis, S. gordonii, and F. nucleatum individually, pairwise and all together. This will include global protein modification analysis using data mining techniques, and a chemical approach to global phosphorylation specifically. We will define protein relative abundance changes (relative to the single organism in isolation) that can be attributed to community microbial interactions. We will thus establish the proteome of the P. gingivalis-F. nucleatum-S. gordonii community, and the differential responses to community development in comparison to individual proteomes. Specific Aim 2 will define the effects of gingival epithelial cells on the P. gingivalis-F. nucleatum-S. gordonii community. This will involve running the same experiments as in Aim 1, but in the presence of epithelial cells, also with comparative posttranslational modification (PTM) analysis on the microbial side. This will define the response of the community to the presence of host cells. As in Aim 1, this includes mining the datasets globally for PTMs for all known modifications using computational approaches, and in addition using a chemically based experimental approach to measuring differential global phosphorylation. In Aim 3 we will prioritize the regulated proteins identified in Aims 1 and 2, as discovery warrants, for additional corroboration and for construction of mutants in the corresponding genes. The phenotype of the mutants in community development and responses to epithelial cells will further elucidate functional meaning and biological relevance.